The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.